Microsatellite loci of genomic DNA have been analyzed for a wide variety of applications, including, but not limited to, paternity testing, forensics work, and in the detection and diagnosis of cancer. Cancer can be detected or diagnosed based upon the presence of instability at particular microsatellite loci that are unstable in one or more types of tumor cells.
A microsatellite locus is a region of genomic DNA with simple tandem repeats that are repetitive units of one to five base pairs in length. Hundreds of thousands of such microsatellite loci are dispersed throughout the human genome. Microsatellite loci are classified based on the length of the smallest repetitive unit. For example, loci with repetitive units of 1 to 5 base pairs in length are termed “mono-nucleotide”, “di-nucleotide”, “tri-nucleotide”, “tetra-nucleotide”, and “penta-nucleotide” repeat loci, respectively.
Each microsatellite locus of normal genomic DNA for most diploid species, such as genomic DNA from mammalian species, consists of two alleles at each locus. The two alleles can be the same or different from one another in length and can vary from one individual to the next. Microsatellite alleles are normally maintained at constant length in a given individual and its descendants; but, instability in the length of microsatellites has been observed in some tumor types (Aaltonen et al., 1993, Science 260:812-815; Thibodeau et al., 1993 Science 260:816-819; Peltomaki et al., 1993 Cancer Research 53:5853-5855; Ionov et al., 1993 Nature 363:558-561). This form of genomic instability in tumors, termed microsatellite instability (hereinafter, “MSI”), is a molecular hallmark of the inherited cancer syndrome Hereditary Nonopolyposis Colorectal Cancer (hereinafter, “HNPCC”). The cause of MSI in HNPCC is thought to be a dysfunctional DNA mismatch repair system that fails to reverse errors that occur during DNA replication (Fishel et al., 1993 Cell 75:1027-38; Leach et al., 1993 Cell 75:215-25; Bronner et al., 1994 Nature 368:258-61; Nicolaides et al., 1994 Nature 371:75-80; Miyaki et al., 1997 Nat Genetics 17:271-2). Insertion or deletion of one or more repetitive units during DNA replication persists without mismatch repair and can be detected as length polymorphisms by comparison of allele sizes found in microsatellite loci amplified from normal and tumor DNA samples (Thibodeau et al, 1993, supra).
MSI has been found in over 90% of HNPCC and in 10-20% of sporadic colorectal tumors (Liu et al., 1996 Nature Med 2:169-174; Thibodeau et al., 1993, supra; Ionov et al., 1993 Nature 363:558-561; Aaltonen et al., 1993 Science 260: 812-816; Lothe et al., 1993 Cancer Res. 53: 5849-5852). However, MSI is not limited to colorectal tumors. MSI has also been detected in pancreatic cancer (Han et al., 1993 Cancer Res 53:5087-5089) gastric cancer (Id.; Peltomaki et al., 1993 Cancer Res 53:5853-5855; Mironov et al., 1994 Cancer Res 54:41-44; Rhyu et al., 1994 Oncogene 9:29-32; Chong et al., 1994 Cancer Res 54:4595-4597), prostate cancer (Gao et al., 1994 Oncogene 9:2999-3003), endometrial cancer (Risinger et al., 1993 Cancer Res 53:5100-5103; Peltomaki et al., 1993 Cancer Res 53:5853-5855), and breast cancer (Patel et al., 1994 Oncogene 9:3695-3700).
The genetic basis of HNPCC is thought to be a germ-line mutation in one of several DNA mismatch repair genes (hereinafter “MMR”) (Leach et al., 1993 Cell 75:1215-1225; Fishel et al., 1993 Cell 75:1027-38; Leach et al., 1993 Cell 75:215-25; Bronner et al., 1994 Nature 368:258-61; Nicolaides et al., 1994 Nature 371:75-80; Miyaki et al., 1997 Nat Genetics 17:271-2; Papadopoulos et al., 1994 Science 263:1625-1629) Among HNPCC patients, 50-60% have been reported to carry inherited mutations in two mismatch repair genes, MSH2 and MLH1 (Kolodner et al., 1999 Cancer Research 59:5068:5074). Moreover, 70-100% of HNPCC cases whose tumors manifest a high frequency MSI (hereinafter “MSI-H”) phenotype reportedly have germ-line mutations in these two genes. Few germ-line mutations in MSH6, MSH3, PMS1 and PMS2 genes have been reported in HNPCC patients, indicating that inherited mutations in these mismatch repair genes play a minor role in HNPCC (Peltomaki et al., 1997 Gastroenterologly 113:1146-1158; Liu et al., 1996 Nat Med 2:169-174; Kolodner et al., 1999 Cancer Research 59:5068-5074). Without functional repair proteins, errors that occur during replication are not repaired leading to high mutation rates and increased likelihood of tumor development.
Repetitive DNA is particularly sensitive to errors in replication and therefore dysfunctional mismatch repair systems result in widespread alterations in microsatellite regions. A study of yeast cells without functional mismatch repair systems showed a 2800, 284, 52, and 19 fold increase in mutation rates in mono-, di-, tri-, tetra-, and penta-nucleotide repeats, respectively (Sia et al., 1997 Molecular and Cellular Biology 17:2851-2858). Mutations in mismatch repair genes are not thought to play a direct role in tumorigenesis, but rather act by allowing DNA replication errors to persist. Mismatch repair deficient cells have high mutation rates and if these mutations occur in genes involved in tumorigenesis the result can lead to the development of cancer. MSI positive tumors have been found to carry somatic frameshift mutations in mono-nucleotide repeats in the coding region of several genes involved in growth control, apoptosis, and DNA repair (e.g., TGFBRII, BAX, IGFIIR, TCF4, MSH3, MSH6) (Planck et al., 2000 Genes, Chromosomes & Cancer 29:33-39; Yamamoto et al., 1998 Cancer Research 58:997-1003; Grady et al., 1999 Cancer Research 59:320-324; Markowitz et al., 1995 Science 268:1336-1338; Parsons et al., 1995 Cancer Research 55:5548-5550). The most commonly altered locus is TGFBRII, in which over 90% of MSI-H colon tumors have been found to contain a mutation in the 10 base polyadenine repeat present in the gene (Markowitz et al., 1995 Science 268:1336-1338).
MSI occurs in almost all HNPCC tumors regardless of which mismatch repair gene is involved. MSI has also been shown to occur early in tumorigenesis. These two factors contribute to making MSI analysis an excellent diagnostic test for the detection of HNPCC. In addition, MSI analysis can serve as a useful pre-screening test to identify potential HNPCC patients for further genetic testing. MSI analysis of sporadic colorectal carcinomas is also desirable, since the occurrence of MSI correlates with a better prognosis (Bertario et al., 1999 International J Cancer 80:83-7).
One long-standing problem with diagnosing HNPCC is that colon tumor biopsies from a person with HNPCC look the same pathologically as a sporadic colon tumor, making diagnosis of the syndrome difficult. Since prognosis, therapy and follow-up will be different for patients with HNPCC, it is important to find more definitive diagnostic methods. However, mutation detection in HNPCC patients remains difficult because there are at least 5 known MMR genes which are large genes without known hot spots for mutations. Direct gene sequencing remains the most precise method of mutation detection, but is time consuming and expensive (Terdiman et al., 1999 The American Journal of Gastroenterology 94:23544-23560). In addition, high sensitivity and specificity can be difficult to obtain with sequencing alone because many mutations that are detected may be harmless polymorphisms that have no affect on the function of the mismatch repair proteins.
DNA analysis of microsatellite loci makes it theoretically possible to develop a blood test for use in the detection of specific types of cancer. Early studies have shown that tumor DNA is released into the circulation, and is present in particularly high concentrations in plasma and serum in a number of different types of cancer (Leon et al., 1977 Cancer Res 37:646-650; Stroun et al., 1989 Oncology 46:318-322). Since then, DNA released into the blood from several different types of tumors has been detected by analysis of microsatellite DNA using the polymerase chain reaction (hereinafter, “PCR”) (Hibi et al., 1998 Cancer Research 58:1405-1407; Chen et al., 1999 Clinical Cancer Research 5:2297-2303; Kopreski et al., 1999 Clinical Cancer Research 5:1961-1965; Fujiwara et al., 1999 Cancer Research 59:1567-1571; Chen et al., 1996 Nature Medicine 2:1033-1034; Goessl et al., 1998 Cancer Research 58:4728-4732; Miozzo et al., 1996 Cancer Research 56:2285-2288).
The first tumor-specific gene sequences detected in blood from patients with cancer were mutated K-ras genes (Vasioukhin et al., 1994 Br. J. Haematol 86: 774-779; Sorenson et al., 1994 Cancer Epidemiol. Biomark. Prev. 3:67-71; Sorenson et al., 2000 Clinical Cancer Research 6:2129-2137; Anker et al., 1997 Gastroenterology 112:1114-1120). More recently, detection of microsatellite instability in soluble tumor DNA from plasma and serum originating from head and neck squamous cell cancers (Nawroz et al., 1996 Nature Med 2:1035-1037) and small cell lung cancers (Chen et al., 1996 Nature Med 2:1033-1035) has been shown. These successes have stimulated searches for microsatellite instability in circulating tumor DNA from many other cancer types. Hibi et al., used microsatellite markers to search for the presence of genetic alterations in serum DNA from colon cancer patients (Hibi, K. et al., 1998 Cancer Research 58:1405-1407). Hibi et al., also reported that eighty percent of primary tumors in the colon cancer patients displayed MSI and/or loss of heterozygosity (hereinafter, “LOH”), another type of mutation discussed below. No microsatellite or LOH mutations were detected in paired serum DNA. However, identical K-ras mutations were found in corresponding tumor and serum DNAs, indicating that tumor DNA was present in the blood. (Id.)
The detection of circulating tumor cells and micrometastases may also have important prognostic and therapeutic implications. Because disseminated tumor cells are present in very small numbers, they are not easily detected by conventional immunocytological tests, which can only detect a single tumor cell among 10,000 to 100,000 normal cells (Ghoussein et al., 1999 Clinical Cancer Research 5:1950-1960). More sensitive molecular techniques based on PCR amplification of tumor-specific abnormalities in DNA or RNA have greatly facilitated detection of occult (hidden) tumor cells. PCR-based tests capable of routinely detecting one tumor cell in one million normal cells have been devised for identification of circulating tumor cells and micrometastases in leukemias, lymphomas, melanoma, neuroblastoma, and various types of carcinomas. (Id.)
Most targets for detection of disseminated tumor cells have been mRNAs. However, some DNA targets have been used successfully, including K-ras mutations in colon cancers, as noted above. The presence of microsatellite instability in some types of tumor cells raises the possibility that these tumor specific mutations created by the instability could serve as a target for PCR-based detection of occult tumor cells.
There has been considerable controversy about how to precisely define and accurately measure MSI (Boland, 1998 Cancer Research 58:5248-5257). Reports on the frequency of MSI in various tumors ranges considerably. For example, different studies have reported ranges of 3% to 95% MSI for the frequency of MSI in bladder cancer (Gonzalez-Zulueta et al., 1993 Cancer Research 53:28-30; Mao et al., 1996 PNAS 91:9871-9875). One problem with defining MSI is that it is both tumor specific and locus dependent (Boland et al. 1998, supra). Thus, the frequency of MSI observed with a particular tumor type in a single study will depend on the number of tumors analyzed, the number of loci investigated, how many loci need to be altered to score a tumor as having MSI and which particular loci were included in the analysis. To help resolve these problems, the National Cancer Institute sponsored a workshop on MSI to review and unify the field (Id.). As a result of the workshop a panel of five microsatellites was recommended as a reference panel for future research in the field. This panel included two mono-nucleotide loci BAT-25, BAT-26, and three dinucleotide loci D5S346, D2S123, D17S250.
One particular problem in MSI analysis of tumor samples occurs when one of the normal alleles for a given marker is missing due to LOH, and no other novel fragments are present for that marker (Id.). One cannot easily discern whether this represents true LOH or MSI in which the shifted allele has co-migrated with the remaining wild-type allele. In cases like this, the recommendation from the NCI workshop on MSI was not to call it as MSI. One way to minimize this type of problem would be to use loci that displayed low frequency of LOH in colon tumors.
Clinical diagnostic assays used for determining treatment and prognosis of disease require that the tests be highly accurate (low false negatives) and specific (low false positive rate). Many informative microsatellite loci have been identified and recommended for MSI testing (Boland et al. 1998, supra). However, even the most informative microsatellite loci are not 100% sensitive and 100% specific. To compensate for the lack of sensitivity using individual markers, multiple markers can be used to increase the power of detection. The increased effort required to analyze multiple markers can be offset by multiplexing. Multiplexing allows simultaneous amplification and analysis of a set of loci in a single tube and can often reduce the total amount of DNA required for complete analysis. To increase the specificity of an MSI assay for any given type of cancer, it has been recommended that the panel of five highly informative microsatellite loci identified at the National Institute Workshop (see above) be modified to substitute or add other loci of equal utility (Boland et al. 1998, supra, at p. 5250). Increased information yielded from amplifying and analyzing greater numbers of loci results in increased confidence and accuracy in interpreting test results.
Multiplex MSI analysis solves problems of accuracy and discrimination of MSI phenotypes, but the additional complexity can make analysis more challenging. For example, when microsatellite loci are co-amplified and analyzed in a multiplex format, factors affecting ease and accuracy of data interpretation become much more essential. One of the primary factors affecting accurate data interpretation is the amount of stutter that occurs at microsatellite loci during PCR (Bacher & Schumm, 1998 Profiles in DNA 2:3-6; Perucho, 1999 Cancer Research 59:249-256). Stutter products are minor fragments produced by the PCR process that differ in size from the major allele by multiples of the core repeat unit. The amount of stutter observed in microsatellite loci tends to be inversely correlated with the length of the core repeat unit. Thus, stutter is most severely displayed with mono- and di-nucleotide repeat loci, and to a lesser degree with tri-, tetra-, and penta-nucleotide repeats (Bacher & Schumm, 1998, supra). Use of low stutter loci in multiplexes would greatly reduce this problem. However, careful selection of loci is still necessary in choosing low stutter loci because percent stutter can vary considerably even within a particular repeat type (Micka et al., 1999 Journal of Forensic Sciences 44:1-15).
Microsatellite multiplex systems have been primarily developed for use in genotyping, mapping studies and DNA typing applications. These multiplex systems are designed to allow co-amplification of multiple microsatellite loci in a single reaction, followed by detection of the size of the resulting amplified alleles. For DNA typing analysis, the use of multiple microsatellite loci dramatically increases the matching probability over a single locus. Matching probability is a common statistic used in DNA typing that defines the number of individuals you would have to survey before you would find the same DNA pattern as a randomly selected individual. For example, a four locus multiplex system (GenePrint™ CTTv Multiplex System, Promega) has a matching probability of 1 in 252.4 in African-American populations, compared to an eight locus multiplex system (GenePrint™ PowerPlex™ 1.2 System, Promega) which has a matching probability of 1 in 2.74×108 (Proceedings: American Academy of Forensic Sciences (Feb. 9-14, 1998), Schumm, James W. et al., p. 53, B88; Id. Gibson, Sandra D. et al., p. 53, B89; Id., Lazaruk, Katherine et al., p. 51, B83; Sparkes, R. et al., 1996 Int J Legal Med 109:186-194). Other commercially available multiplex systems for DNA typing include AmpFlSTR Profiler™ and AmpFlSTR COfiler™ (AmpFlSTR Profiler™ PCR Amplification Kit User's Manual (1997), i-viii and 1-1 to 1-10; and AmpFlSTR COfiler™ PCR Amplification Kit User Bulletin (1998), i-iii and 1-1 to 1-10, both published by Perkin-Elmer Corp). In addition to multiplexes for DNA typing, a few multiplex microsatellite systems have been developed for the detection of diseases, such as cancer. One such system has been developed by Roche Diagnostics, the “HNPCC Microsatellite Instability Test”, in which five MSI loci (BAT25, BAT26, D5S436, D17S250, and D2S123) are co-amplified and analyzed. Additional systems are needed, particularly systems that include additional loci displaying high sensitivity to MSI and low stutter for easy and accuracy of analysis.
The materials and methods of the present invention are designed for use in multiplex analysis of particular microsatellite loci of human genomic DNA from various sources, including various types of tissue, cells, and bodily fluids. The present invention represents a significant improvement over existing technology, bringing increased power of discrimination, precision, and throughput to the analysis of MSI loci and to the diagnosis of illness, such as cancer, related to mutations at such loci.